Bacterial biofilms are associated with the persistent infections because of their high tolerance to antimicrobial agents. Hence, controlling pathogenic biofilm formation is important in bacteria-related diseases. Staphylococcus aureus is a versatile human pathogen that readily forms biofilms on human tissues and diverse medical devices. As S.aureus can be naturally found in multi-species communities, the supernatants of 28 bacteria were screened to identify new biofilm inhibitory components against S.aureus. The culture supernatant (1%, v/v) of Pseudomonas aeruginosa PAO1 inhibited S.aureus biofilm formation more than 90% without affecting its planktonic cell growth. The P.aeruginosa supernatant contained a high protease activity, which both inhibited S.aureus biofilm formation and detached pre-existing biofilms. An examination of 13 protease-deficient P.aeruginosa mutants identified that LasB elastase is a major antibiofilm protease in P.aeruginosa against S.aureus. Transcriptional analyses showed that P.aeruginosa supernatant induced the expression of endogenous protease genes (aur, clp, scpA, splA, and sspA) and other regulatory genes (agrA, hla, and saeS). Additionally, exogenous proteinase K clearly enhanced the protease activity of S.aureus. Hence, S.aureus accelerated the expression of its own protease genes in the presence of exogenous protease, leading to the rapid dispersal of its biofilm.